Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
Environmental stresses, such as drought, increased salinity of soil, and extreme temperature, are major factors in limiting plant growth and productivity. The worldwide loss in yield of three major cereal crops, rice, maize (corn), and wheat due to water stress (drought) has been estimated to be over ten billion dollars annually. Breeding of stress-tolerant crop cultivars represents a promising strategy to tackle these problems (Epstein et al., 1980). However, conventional breeding is a slow process for generating crop varieties with improved tolerance to stress conditions. Limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species are additional problems encountered in conventional breeding. Recent progress in plant genetic transformation and availability of potentially useful genes characterized from different sources make it possible to generate stress-tolerant crops using transgenic approaches (Tarczynski et al., 1993; Pilon-Smits et al., 1995).
Characterization and cloning of plant genes that confer stress tolerance remains a challenge. Genetic studies revealed that tolerance to drought and salinity in some crop varieties is principally due to additive gene effects (Akbar et al., 1986a, 1986b). However, the underlying molecular mechanism for the tolerance has never been revealed. Physiological and biochemical responses to high levels of ionic or nonionic solutes and decreased water potential have been studied in a variety of plants. Based on accumulated experimental observations and theoretical consideration, one suggested mechanism that may underlie the adaptation or tolerance of plants to osmotic stresses is the accumulation of compatible, low molecular weight osmolytes such as sugar alcohols, special amino acids, and glycinebetaine (Greenway and Munns, 1980; Yancey et al., 1982). Recently, a transgenic study has demonstrated that accumulation of the sugar alcohol mannitol in transgenic tobacco conferred protection against salt stress (Tarczynski et al., 1993). Two recent studies using a transgenic approach have demonstrated that metabolic engineering of the glycinebetaine biosynthesis pathway is not only possible but also may eventually lead to production of stress-tolerant plants (Holmstrom et al., 1994; Rathinasabapathi et al., 1994).
In addition to metabolic changes and accumulation of low molecular weight compounds, a large set of genes is transcriptionally activated which leads to accumulation of new proteins in vegetative tissue of plants under osmotic stress conditions (Skriver and Mundy, 1990; Chandler and Robertson, 1994). The expression levels of a number of genes have been reported to be correlated with desiccation, salt, or cold tolerance of different plant varieties of the same species. It is generally assumed that stress-induced proteins might play a role in tolerance, but direct evidence is still lacking, and the functions of many stress-responsive genes are unknown. Elucidating the function of these stress-responsive genes will not only advance our understanding of plant adaptation and tolerance to environmental stresses, but also may provide important information for designing new strategies for crop improvement (Chandler and Robertson, 1994).
Late embryogenesis abundant proteins (LEA proteins) were first characterized in cotton as a set of proteins that are highly accumulated in the embryos at the late stage of seed development (Dure et al., 1981). Subsequently, many LEA proteins or their genes have been characterized from different plant species (collated by Dure, 1992). Based on their common amino acid sequence domains, LEA proteins were classified into three major groups (Baker et al., 1988; Dure et al., 1989). A group 2 LEA protein and its cDNA have been characterized from rice (Mundy and Chua, 1988). The four members of a group 2 LEA gene family are tandemly arranged in a single locus, and are coordinately expressed in various rice tissues in response to ABA, drought, and salt stress (Yamaguchi-Shinozaki et al., 1989). However, the functions of these LEA proteins are unknown. Recently, both group 2 and group 3 LEA proteins have been characterized from Indica rice varieties and the accumulation of these LEA proteins in response to salt stress were correlated with varietal tolerance to salt stress (Moons et al., 1995). Group 2 LEA proteins (dehydrins) containing extensive consensus sequence were detected in a wide range of plants, both monocots and dicots (Close et al., 1993). A recent study showed that a group 2 LEA gene is present in many plant species but the expression of this gene is differentially regulated in stress sensitive and tolerant species (Danyluk et al., 1994).
A barley group 3 LEA protein, HVA1, was previously characterized from barley aleurone. The HVA1 gene is specifically expressed in the aleurone layers and the embryos during late stage of seed development, correlating with the seed desiccation stage (Hong et al., 1988). Expression of the HVA1 gene is rapidly induced in young seedlings by ABA and several stress conditions including dehydration, salt, and extreme temperature (Hong et al., 1992).
HVA1 protein belongs to the group 3 LEA proteins that include other members such as wheat pMA2005 (Curry et al., 1991; Curry and Walker-Simmons, 1993), cotton D-7 (Baker et al., 1988), carrot Dc3 (Seffens et al., 1990), and rape pLEA76 (Harada et al., 1989). These proteins are characterized by 11-mer tandem repeats of amino acid domains which may form a probable amphophilic alpha-helical structure that presents a hydrophilic surface with a hydrophobic stripe (Baker et al., 1988; Dure et al., 1988; Dure, 1993). The barley HVA1 gene and the wheat pMA2005 gene (Curry et al., 1991; Curry and Walker-Simmons, 1993) are highly similar at both the nucleotide level and predicted amino acid level. These two monocot genes are closely related to the cotton D-7 gene (Baker et al., 1988) and carrot Dc3 gene (Seffens et al., 1990) with which they share a similar structural gene organization (Straub et al., 1994).
In many cases, the timing of LEA mRNA and protein accumulation is correlated with the seed desiccation process and associated with elevated in vivo abscisic acid (ABA) levels. The expression of LEA genes is also induced in isolated immature embryos by ABA, and in vegetative tissues by ABA and various environmental stresses, such as drought, salt, and extreme temperature (Skriver and Mundy, 1990; Chandler and Robertson, 1994).
There is, therefore, a correlation between LEA gene expression or LEA protein accumulation with stress tolerance in a number of plants. For example, in severely dehydrated wheat seedlings, the accumulation of high levels of group 3 LEA proteins was correlated with tissue dehydration tolerance (Ried and Walker-Simmons, 1993). Studies on several Indica varieties of rice showed that the levels of group 2 LEA proteins (also known as dehydrins) and group 3 LEA proteins in roots were significantly higher in salt-tolerant varieties compared with sensitive varieties (Moons et al., 1995).
On the other hand, the presence of other LEA proteins is not always correlated with stress tolerance. For example, comparative studies on wild rice and paddy rice showed that the intolerance of wild rice seeds to dehydration at low temperature is not due to an absence of or an inability to synthesize group 2 LEA/dehydrin proteins, ABA, or soluble carbohydrates (Bradford and Chandler, 1992; Still et al., 1994). Overproduction of a group 2 LEA protein from the resurrection plant Craterostigma in tobacco did not confer tolerance to osmotic stress (Iturriaga et al., 1992). It has been found that LEA proteins are not sufficient to confer desiccation tolerance in soybean seeds, and it is the LEA proteins together with soluble sugars that contribute to the tolerance (Blackman et al., 1991, 1992).
In these reported cases of increased water stress or salt stress tolerance, a large set of genes has been activated in the stressed plant (Skriver and Mundy, 1990; Chandler and Robertson, 1994). The LEA protein(s) are the product of just one of these gene(s), and many other proteins are also correlated with the increased water stress or salt stress tolerance (i.e. levels of these other proteins also increase in response to water stress or salt stress). Therefore, although a correlation exists between LEA proteins and increased water stress or salt stress tolerance, no evidence exists that any of the particular activated genes (including the LEA genes) can confer water stress or salt stress tolerance upon a plant. Accordingly, identification of appropriate genes for use in genetic engineering of plants to increase water stress or salt stress tolerance has not been attained.
A need exists, therefore, for the identification of a gene encoding a protein that can confer water stress or salt stress tolerance on a plant transformed with the gene. Such a water stress or salt stress tolerant plant can find many uses, particularly in agriculture and particularly in regard to cereal plants which are a major crop plant.